Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates

Background The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. Results Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6’)-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. Conclusions Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates Sepideh Rezaei 1,2 , Saeed Tajbakhsh 1 , Behrouz Naeimi 1 and Forough Yousefi 1* Background Fluoroquinolones (FQs) are commonly used as effective antibiotics for the treatment of most infections caused by Gram-negative bacteria [1].Unfortunately, due to their extensive use, FQ resistance (FQ-R) is on the rise in clinically important bacteria, including Klebsiella pneumoniae [1,2].FQs inhibit the activity of DNA gyrase and topoisomerase IV enzymes, which are essential enzymes for bacterial DNA replication and survival [2].The main mechanisms of resistance to FQs in Enterobacteriaceae arise from chromosomal mutations in the quinolone resistance-determining region (QRDR), particularly of gyrA and parC encoding DNA gyrase, and topoisomerase IV, respectively.These mutations lead to structural changes in DNA gyrase and/or topoisomerase IV, which impairs the affinity to FQs [3].Plasmid-mediated quinolone resistance (PMQR) genes are alternative mechanism of quinolone resistance.However, PMQR has been shown to emerge even in the absence of FQ therapy [4].Three types of PMQR determinants have been identified in Enterobacteriaceae: (i) Qnr proteins, encoded by qnr genes (qnrA, qnrB, qnrC, qnrD and qnrS), belong to the pentapeptide repeat family and protect DNA gyrase and topoisomerase IV from quinolone inhibition by binding to them [1,5].(ii) The AAC(6')-Ib-cr enzyme, a variant aminoglycoside acetyltransferase encoded by aac(6')-Ib-cr gene, can modify ciprofloxacin with a piperazinyl substituent, reducing its activity [6].(iii) OqxAB and QepA pumps reduce susceptibility to FQs by drug extrusion from the cell.These multidrug efflux pumps belong to the resistance-nodulation-cell division (RND) family and the major facilitator superfamily (MFS), respectively [2,7].The patterns of resistance mechanisms to FQ vary across different countries due to geographical impact on the emergence and dissemination of FQ-R mechanisms [1].Thus, it is important to determine the major FQ-R mechanisms in each geographical area.The aim of this study was to determine chromosomal mutations in gyrA and parCgenes as well as the prevalence of PMQR genes among fluoroquinolone resistant K. pneumoniae isolates in Bushehr province, Iran.

Bacterial isolation and identification
This project was approved by the Ethical Committee of Bushehr University of Medical Sciences with reference number IR.BPUMS.REC.1400.133.A total of 215 K. pneumoniae isolates were collected from six hospitals located in Bushehr province, in the south of Iran from November 2017 to February 2019.Bacterial identification was conducted by biochemical tests and confirmed by PCR to target malate dehydrogenase (mdh), the genusspecific housekeeping gene (Table 1) [8][9][10].

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was determined by disk diffusion method for ciprofloxacin (5 mg) and levofloxacin (5 mg) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [18].In addition, MIC of ciprofloxacin was determined using E-tests (Liofilchem, Italy) on Mueller-Hinton agar (Biolife, Italy).Escherichia coli ATCC 25922 was used as the control strain for the antibiotic susceptibility tests.

Detection of mutations in QRDR of gyrA and parC genes
A total of 40 ciprofloxacin-resistant isolates were selected.PCR amplification of gyrA and parC genes were carried out using primers and conditions listed in Table 1.A total reaction volume of 25 µl contained 12.5 µl 2x MasterMix (Ampliqon, Odense, Denmark), 1 µl (10 µmol) of each forward and reverse primer, 1 µl of template DNA; and 9.5 µl of nuclease-free water.The amplification conditions were as follows: pre-denaturation at 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C/59°C (gyrA / parC) for 30 s, and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 5 min.Nucleotide sequencing of the PCR products was performed by the Bioneer Company (Seoul, Korea).Mutations in gyrA and parC genes for the 40 ciprofloxacin-resistant K. pneumoniae isolates were compared with the reference sequences of gyrA gene of K. pneumoniae ATCC13883 (GenBank accession number: DQ673325) and parC gene of K. pneumoniae ATCC1388T (GenBank accession number: AF303641).
Online sequence alignment and analysis were performed using the online ClustalW2 multiple sequence alignment program.

Nucleotide sequence accession number
The sequences of detected genes were submitted to the GenBank database under accession numbers OQ281591 -OQ281600.

Correlation of ciprofloxacin MIC values with mutations in the QRDRs of gyrA and parC genes
Notably, a significant correlation between the frequency of mutations in QRDR of gyrA and parC and ciprofloxacin MIC values was observed in the present study, as the results showed that 29 out of 31 isolates in which the MIC of ciprofloxacin was ≥ 32 µg/ml had 2 or 3 mutations in both gyrA and parC genes simultaneously, while in 4 resistant isolates in which the MIC of ciprofloxacin was equal to 4 µg/ml, two isolates had no mutations and the other two isolates had only one mutation in the gyrA gene.It is worth noting that there was no correlation between the MIC values of ciprofloxacin and the number of harbored PMQR genes.

Discussion
Fluoroquinolones, such as ciprofloxacin, are a group of effective drugs for the treatment of Klebsiella pneumoniae infections, which inhibit bacterial DNA replication by binding to topoisomerase IV and DNA gyrase enzymes [19].Unfortunately, resistance to these antimicrobial agents has emerged, and the level of resistance is increasing due to their widespread use.
The main mechanism of resistance to FQs in bacteria is spontaneous mutations in the QRDR of gyrA gene encoding DNA gyrase and parC gene encoding topoisomerase IV, particularly at the highly conserved residues Ser83 and Asp87 of gyrA gene [20].
In the present study, we investigated mutations in the QRDR of gyrA and parC genes and the prevalence of PMQR genes among fluoroquinolone resistant K. pneumoniae clinical isolates.
Although mutations in QRDR of gyrA and parC genes are the main cause of FQs resistance, PMQR genes contribute to fluoroquinolone resistance due to their high horizontal transferability [21].
Our molecular analysis identified that 40 (100%) ciprofloxacin-resistant isolates harbored at least one PMQR determinant and the most common PMQR gene among our isolates was oqxA gene (100%).Prevalence of PMQR genes in the study conducted by Jomehzadeh et al. [21] was 88% and aac(6')-Ib-cr was detected as the most common PMQR gene (50%), while in another study done by Sani et al. 85.4% of the isolates harbored PMQR genes and the most prevalent PMQR gene was qnrS (41.67%) [26].Furthermore, qnrA and qepA genes have not been reported in several studies conducted in some geographic areas, including Korea, Malaysia, and Iran which support our data for these genes [27,28].
The main mechanism of resistance to FQ in the Enterobacteriaceae is alterations in QRDR of gyrA, which encodes DNA gyrase, a type II topoisomerase [29].
Mutation analysis in QRDR of gyrA gene revealed that 35(87.5%)out of 40 ciprofloxacin-resistant isolates had at least one mutation in gyrA gene and the most frequent amino acid substitution in gyrA gene was Ser83→Ile, which was found in 52.5% of our isolates.This finding was inconsistent with the studies done by   [29].In the study conducted by Azargun et al., Ser83→Leu plus Asp87→Asn double mutations were detected in 60% of FQ-resistant K. pneumoniae isolates [2].In agreement with their findings, one of the five double mutations identified in our study was Ser83→Phe plus Asp87→Asn but the four remaining double mutations of the present study were not found in the Fu, and Azargun's studies.Therefore, the effect of the other double mutations was ignored in the mentioned studies.It is notable that, in agreement with the present study, the most common double mutation observed in the Akya's study was Ser83→Phe plus Asp87→Ala, which was present in 42.9% of FQ-resistant K. pneumoniae isolates [27].Moreover, similar to the current study Ser83→Phe plus Asp87→Asn double mutations were detected in 14.3% of FQ-resistant K. pneumoniae isolates.
However, the three remaining double mutations of the present study were not found in their study.
In addition, in our study 14 (35%) isolates had three mutations in QRDR of both gyrA and parC genes.Notably, the frequency of mutations in QRDR of gyrA and parC revealed a significant effect on ciprofloxacin MIC values; as the results showed 29 isolates out of 31 isolates in which the MIC of ciprofloxacin was ≥ 32 µg/ml had 2 or 3 mutations in both gyrA and parC genes simultaneously, while in 4 resistant isolates in which the MIC of ciprofloxacin was equal to 4 µg/ml, two isolates had no mutations and the other two isolates had only one mutation in the gyrA gene.

Conclusion
In conclusion, all ciprofloxacin-resistant K. pneumoniae isolates either had mutations in the QRDR of gyrA and parC genes or carried PMQR genes.Our results showed 90% of ciprofloxacin-resistant K. pneumoniae isolates had at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutation in QRDR was very significant and alarming in our region.Amino acid substitution Ser83→Ile in gyrA which has the greatest impact on ciprofloxacin resistance and Ser80→Ile in parC genes were the most frequent mutations among our FQ-R K. pneumoniae isolates.In addition, acquisition of 2 or 3 mutations in both gyrA and parC genes played an important role in conferring high level resistance to ciprofloxacin.

Table 1
Oligonucleotide primers used for detection PMQR determinants and mutation in QRDR

Table 3
The frequency of mutations and amino acid changes within gyrA, and parC of 40 ciprofloxacin -resistant isolates WT: wild type